Familial hypertrophic cardiomyopathy (FHC) is one of the pathological compensatory manifestations found in the heart resulting from its inability to adequately pump blood, thus leading to hypertrophy and often to premature cardiac death. Over the past 4 years our laboratory has been studying the functional consequences of several FHC mutations in the regulatory light chain (RLC) of myosin expressed in transgenic mice. We hypothesize that by changing the properties of the RLC Ca2+-Mg2+ binding site, the FHC mutations interfere with the intracellular function of RLC as a temporary delayed Ca2+- buffer and lead to increased or decreased kinetics of muscle relaxation. Another hypothesis pertains to the mutation controlled metal occupancy of the Ca2+-Mg2+ binding site of RLC and the mechanism by which Ca2+ or Mg2+ binding to RLC may influence the interaction of myosin with actin and tension generation. We further hypothesize that an FHC induced pathological cardiac phenotype can be rescued by Ca2+-calmodulin activated MLCK phosphorylation of the RLC-mutated myocardium. This application will continue the use of integrated multidisciplinary approaches from single molecule, cell to organ levels and novel transgenic mouse models to address the following questions: SPECIFIC AIM 1: Do FHC induced changes in the properties of the RLC Ca2+-Mg2+ binding site inhibit or facilitate the function of RLC as a temporary intracellular calcium buffer? Do FHC mutations shift the metal occupancy of the RLC Ca2+-Mg2+ binding site during muscle contraction? SPECIFIC AIM 2: Is RLC phosphorylation by Ca2+-calmodulin (CaM) activated myosin light chain kinase (MLCK) affected by FHC-linked RLC mutations? Can MLCK phosphorylation rescue a mutation induced pathological cardiac phenotype? SPECIFIC AIM 3: Do FHC-associated mutations in RLC alter intermolecular interactions between RLC and myosin heavy chain (HC) and ultimately myosin and actin? Do these changes lead to myofilament disarray, cardiac hypertrophy and dysfunction of the mutated myocardium? Successful execution of this proposal will result in new mechanical, physiological and histological information regarding the role of the RLC in cardiac muscle contraction in health and disease. [unreadable] [unreadable] Relevance: Cardiovascular diseases are the number one cause of mortality worldwide with heart failure being highly prevalent in most affluent parts of the world. There is an urgent need for a better understanding of the mechanisms underlying Familial Hypertrophic Cardiomyopathy (FHC) that often leads to premature sudden cardiac death (SCD). This proposal addresses the mechanisms by which the mutations in myosin regulatory light chain (RLC) cause FHC and lead to SCD. Determining the mechanisms of the RLC- mediated regulation of contraction in the healthy and hypertrophic heart will provide insight and be instrumental in developing specific therapeutic strategies that can be employed to reverse or prevent FHC RLC pathology. [unreadable] [unreadable] [unreadable]